Universidad de Buenos...
L-carnitine (LC) plays an important role in lipid catabolism and protects cells from damage caused by reactive oxygen species (ROS) due to its antioxidant activity. The \nobjective of this work was to evaluate if the supplementation of LC to the in vitro maturation medium (IVM) decreases the content...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2023
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7378 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7378.dir/7378.PDF |
| Aporte de: |
| Sumario: | L-carnitine (LC) plays an important role in lipid catabolism and protects cells from damage caused by reactive oxygen species (ROS) due to its antioxidant activity. The \nobjective of this work was to evaluate if the supplementation of LC to the in vitro maturation medium (IVM) decreases the content of intracellular lipids, decreases the \nintracellular level of ROS, favours mitochondrial function and increases the concentration \nof total glutathione (GSH) improving oocyte maturation, embryo quality and cryo-tolerance to vitrification of in vitro matured porcine oocytes. The cumulus-oocyte \ncomplexes were obtained by follicular aspiration of ovaries from sacrificed sows and matured in vitro for 44 h without LC (control) or with different concentrations of LC (0.6,\n1.25 or 2.5 mg/mL) (Sigma-Aldrich) in supplemented TCM-199. In vitro fertilization was performed with fresh boar semen for four h in 100 µL-drop of MCT 199 with caffeine, BSA, sodium lactate and pyruvate (20 denuded oocytes per drop, 1x106 spermatozoa/mL). Presumptive zygotes were washed and cultured at NCSU 23 at 39°C, \n7 % O2, 5 % CO2, and humidity. The percentage of cleavage was recorded on day two, and the percentage of blastocyst on day seven. In order to establish the best \nconcentration of LC to use to vitrify mature porcine oocytes, different determinations \nwere made: percentage of nuclear maturation, intracellular lipid content, intracellular level of ROS, mitochondrial function, apoptosis in cumulus cells (CC), total GSH concentration, post-IVF parameters and embryonic development. The concentration of 0.6 mg/mL of LC significantly decreased the intracellular lipid content, the intracellular \nlevel of ROS, and the mitochondrial function without affecting the percentage of nuclear \nmaturation or the total GSH concentration of the oocytes compared to the control. LC \nduring IVM affected the CC viability of mature porcine oocytes. The concentration of 2.5 mg/mL of LC significantly decreased the nuclear maturation of the oocytes to the control. \nNo significant differences between LC treatments and control were observed in post-IVF parameters (percentage of penetrated and monospermic oocytes, pronucleus formation, \nand efficiency). However, adding 0.6 mg/mL of LC during IVM decreased cleavage rates \nwithout modifying blastocyst rates, cell number, or blastocyst apoptosis compared with \nthe control. Hatched blastocysts were observed upon maturing LC oocytes but were not in control. Based on these previous results, the 0.6 mg/mL concentration of LC was selected to be used during IVM prior to the vitrification of mature oocytes with a new device (alternative vitrification support). LC during IVM decreased the number of dead oocytes after vitrification and tempering compared with the control. However, there were no significant differences in the percentages of early and late apoptosis, the intracellular ROS level and the oocytes' mitochondrial function in vitrified and tempered ripe, either using LC or not during IVM. Vitrification negatively affected porcine oocytes matured or not with LC with a low percentage of morphologically normal MII at 2 and 4 h post-tempering, without normal IVF (assessed by post-IVF parameters), with a low percentage of segmentation in control and null in the treatment with LC and without blastocysts in either of the two treatments. This is the first work in porcine species where the effect of LC is evaluated during IVM prior to the vitrification of mature oocytes. \nTreatment with 0.6 mg/mL of LC improved vitrified oocytes viability. However, neither the antioxidant function of LC nor the decrease in intracellular lipid content of oocytes was sufficient to reverse the adverse effects of cryopreservation of mature porcine oocytes. \nWork should continue on improvements during the vitrification and tempering of mature oocytes for this technique to be viable in porcine species. |
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